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International Journal of Biological Macromolecules 79 (2015) 611–617

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

heological studies of the fucose-rich exopolysaccharide FucoPol

ristiana A.V. Torresa, Ana R.V. Ferreirab, Filomena Freitasa, Maria A.M. Reisa,sabel Coelhosob, Isabel Sousac, Vítor D. Alvesc,∗

UCIBIO-REQUIMTE, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, PortugalLAQV-REQUIMTE, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, PortugalLEAF – Linking Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017, Portugal

r t i c l e i n f o

rticle history:eceived 17 April 2015eceived in revised form 14 May 2015ccepted 18 May 2015vailable online 23 May 2015

eywords:

a b s t r a c t

In this work, the solution properties of the bacterial fucose-rich polysaccharide, FucoPol, were studied. Theeffect of pH (3.5–10.0) and ionic strength (0.02–1.0 M NaCl) on the intrinsic viscosity and steady shearflow were evaluated using a central composite rotatable design of experiments and surface responsemethodology. FucoPol’s intrinsic and apparent viscosities presented a quite low variation under a widerange of pH (3.5–8.0) and ionic strength (0.05–0.50 M NaCl) values. FucoPol produced viscous solutionswith shear-thinning behavior at different polymer concentrations (0.2–1.2 wt.%). Flow curves were suc-

ucose-rich polysaccharideolution properties, Rheology

cessfully described by the Cross model. The viscosity of the first Newtonian plateau varied from 0.01to 2.47 Pa s for polymer concentrations from 0.2 to 1.2 wt.%, and the dependence of the estimated relax-ation time with polymer concentration suggests a large degree of interaction between FucoPol molecules.Given the results obtained, FucoPol is proposed as thickening agent for applications in which stability ofthe apparent viscosity under pH and ionic strength variations is required.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Water soluble polysaccharides are industrially important mate-ials due to their rheological properties in aqueous systems.nderstanding polysaccharides’ properties in aqueous solutions isritical to forecast their potential industrial applications in spe-ific areas, such as food products, cosmetics, pharmaceuticals, oilrilling fluids and paints, being important to the manufacture,istribution, storage and consumption of many products. Theseroperties can be affected by several parameters, such as pH, saltoncentration, temperature, polymer average molecular weightnd shear rate [1]. For some applications, it is important to haveiscous solutions at low concentrations, and stable under a wideange of temperatures, pH and ionic strengths, namely in emulsionsn the food industry [2,3] and in oil drilling fluids [4,5].

Bacterial polysaccharides frequently present distinctive solu-ion properties that are not demonstrated by traditional polymerserived from other natural sources (plants, algae and animals) [6,7].

xamples of commercial bacterial polysaccharides include xanthanum, gellan gum, fucogel, hyaluronic acid and welan gum. Others,ith either none or low commercial expression have been reported,

∗ Corresponding author. Tel.: +351 21 3653546; fax: +351 21 3653200.E-mail address: vitoralves@isa.ulisboa.pt (V.D. Alves).

ttp://dx.doi.org/10.1016/j.ijbiomac.2015.05.029141-8130/© 2015 Elsevier B.V. All rights reserved.

for example colanic acid [8], clavan [9] and rhamsan [10]. Beyondthe solution properties, they may also present interesting biolog-ical activities, namely those composed of l-fucose, l-rhamnose oruronic acids residues. Such functional properties are the drivingforce for the search of new bacterial polysaccharides with potentialto be used on specific applications [6,11].

FucoPol is a fucose-rich polysaccharide synthesized by thebacterium Enterobacter A47. It presents a high molecular weight(5 × 106) and a low polydispersity index (1.3), being com-posed of fucose (32–36 mol%), galactose (25–26 mol%), glucose(28–37 mol%), glucuronic acid (9–10 mol%) and acyl groups, namelysuccinyl (2–3 wt.%), pyruvyl (13–14 wt.%) and acetyl (3–5 wt.%).The purified polymer samples present a protein content below5 wt.% and traces of inorganic salts [12–14]. The presence of glu-curonic acid, as well as pyruvyl and succinyl, confers FucoPola polyelectrolyte character. Previous works have demonstratedthat FucoPol solutions, with a concentration around 1 wt.%, havea shear-thinning behavior and mechanical spectra revealing vis-cous solutions with entangled polymer molecules, in the rangeof temperatures within 15–65 ◦C [12,13]. Moreover, the apparentviscosity and the viscoelastic properties, measured at 25 ◦C, were

maintained after consecutive heating and cooling cycles, indicat-ing a good thermal stability under temperature fluctuations [15].However, additional studies are necessary in order to improvethe knowledge of FucoPol aqueous solutions properties, namely

6 f Biological Macromolecules 79 (2015) 611–617

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2

2

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2

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2

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wrKee

Table 1Central composite rotatable design (CCRD) with two independents variables X1

(Ionic Strength, IS) and X2 (pH), and the observed responses studied Y1 (intrinsicviscosity [�]) and Y2 (zero-shear rate viscosity �0) for 1 wt.% FucoPol solution.

Run number IS (M NaCl) pH [�] (dL/g) �0 (Pa s)X1 X2 Y1 Y2

Factorialdesign

1 0.15 4.50 8.17 0.942 0.65 4.50 7.37 0.883 0.15 9.50 7.38 0.414 0.65 9.50 7.21 0.43

Centralpoint

5 0.40 7.00 8.30 1.206 0.40 7.00 8.28 1.207 0.40 7.00 8.23 1.20

8 0.05 7.00 8.54 0.649 0.75 7.00 5.65 0.61

12 C.A.V. Torres et al. / International Journal o

egarding the effect of different pH, ionic strength and polymeroncentration.

In this work, the solution properties of the fucose-richxopolysaccharide FucoPol were evaluated. The assessment of theolymer’s behavior in aqueous solutions involved the study of (i)he impact of pH and ionic strength on the intrinsic viscosity; (ii)he steady shear behavior, with focus on the effect of polymer con-entration, pH and ionic strength on the apparent viscosity; and (iii)he concentration regimes. These studies are essential to envisageucoPol’s potential applications, since polysaccharides are mainlysed in aqueous formulations, which are the base of many sys-ems, such as drilling fluids, food products, and some cosmeticsnd pharmaceuticals.

. Materials and methods

.1. FucoPol production and extraction

FucoPol was obtained by cultivation of the bacterium Entero-acter A47 (DSM 23139) on mineral medium supplemented withlycerol byproduct from the biodiesel industry, as previouslyeported [12,14]. The culture broth recovered from the bioreac-or at the end of the cultivation was diluted with deionized water1:2, v/v) for viscosity reduction and centrifuged (13 000 × g, 1 h) inrder to remove cells. The cell-free supernatant was subjected torotein thermal denaturation (70 ◦C, 1 h), to avoid enzymatic poly-er degradation during the subsequent purification steps, followed

y their separation by centrifugation (13 000 × g, 1 h). FucoPol-richupernatant was dialysed with a 10 000 MWCO membrane (SnakekinTM Pleated Dialysis Tubing, Thermo Scientific) against deion-zed water, for 48 h at 4 ◦C. Finally, the purified supernatant wasreeze-dried.

.2. FucoPol solutions

FucoPol, purified as described in Section 2.1, was added to NaClolutions or deionized water, depending on the ionic strengthalue used, and stirred overnight at room temperature. FucoPoloncentration ranged from 0.02 to 0.06 wt.% for intrinsic viscos-ty measurements, and from 0.2 to 1.2 wt.% for apparent viscosity

easurements. The solutions’ ionic strength ranged from roughly.05 to 0.075 M NaCl. The pH of the solutions was adjusted to theesired values by addition of small drops of HCl (15 wt.%) and/oraOH (10 wt.%) aqueous solutions.

.3. Intrinsic viscosity measurements

Capillary viscosity measurements were performed using a glassiscometer (Schott Micro-Ubbelohde Viscometer Ic) immersed in

water bath at constant temperature (25 ± 0.5 ◦C). The reducednd inherent viscosities were measured from capillary flow timesorrected for density perturbations, for at least seven FucoPoloncentration values (three replicas for each concentration). Thentrinsic viscosity was calculated by extrapolation to zero concen-ration of Huggins (Eq. (1)) and Kraemer (Eq. (2)) equations:

�sp

C= [�] + KH[�]2c (1)

ln(�rel)C

= [�] + KK[�]2C (2)

here �sp (dL/g), [�] (dL/g) and �rel are the specific, intrinsic and

elative viscosities, respectively. KH and KK are the Huggins andraemer constants, and c (g/dL) is the polymer concentration. Allxperiments were carried out in the range 1.2 < �rel < 2.0 in order tonsure a good accuracy in the extrapolations to zero concentration.

Axialpoints 10 0.40 3.47 8.07 1.07

11 0.40 10.54 5.51 0.37

2.4. Apparent viscosity measurements

The apparent viscosity of FucoPol’s aqueous solutions wasmeasured by loading directly the solutions on a cone and plategeometry (diameter 35 mm, angle 2◦) of a controlled stress rheome-ter (Rheostress RS 75, Haake, Germany). The shearing geometrywas covered with paraffin oil in order to minimize sample dehy-dration. The samples were equilibrated at 25.0 ± 0.1 ◦C, for 10 min,after which the flow curves were obtained using a steady-state flowramp (torque was imposed using a logarithmic ramp) in the shearrate range from 1 to 700 s−1.

2.5. Experimental design

Response surface methodology (RSM) was applied to evaluatethe combined effect of the ionic strength (NaCl concentration) andthe pH, both on the observed intrinsic viscosity [�] (Y1), and onthe zero shear rate viscosity (first Newtonian plateau) (Y2) of solu-tions with a FucoPol concentration of 1 wt.%. A central compositerotatable design (CCRD), with two independent variables, where X1is the ionic strength, IS (M, NaCl concentration), and X2 is the pH(Table 1), was used. The conditions of the central point of the design(NaCl 0.40 M, at pH 7.0) were tested three times, to allow estimat-ing the experimental error. All experiments were carried out on arandomized order to prevent the effect of unexplained variabilitydue to exogenous factors.

The system’s behavior was evaluated by fitting the experimentaldata to the following second order model:

Yp = b +3∑

i=1

aiXi +3∑

i=1

3∑

j=1,j /= i

aijXiXj +3∑

j=1

aiiX2i (3)

where Yp is the predicted response, Xi is the coded value of theindependent variable i; b is the intercept and ai, aij, aii are thelinear, interaction and quadratic coefficients, respectively [16]. Inorder to identify an appropriate reduced quadratic model, the sig-nificance of each source of variation was obtained from statisticalanalysis (ANOVA). The statistical analysis was carried out using anappropriate software.

3. Results

3.1. Solution properties in dilute regime

3.1.1. Polyelectrolyte behavior in deionized water and in 0.1 MNaCl

Fig. 1a presents the variation of the reduced viscosity asa function of FucoPol concentration in a salt free aqueous

C.A.V. Torres et al. / International Journal of Biological Macromolecules 79 (2015) 611–617 613

F(a

sedouiawhiibrf

nAtafeovrx

0c[0a

3

sa

Table 2Analysis of variance of the second order model for parameter [�] (intrinsic viscosity).

Source of variation [�]

Sum ofsquares

Df Meansquared

F-value p-value

IS 5.706 1 5.706 595.128 <0.001pH 1.999 1 1.999 208.478 <0.001IS.IS 5.013 1 5.013 522.783 <0.001pH.pH 3.097 1 3.097 323.038 <0.001IS.pH 0.666 1 0.666 69.510 0.001Lack-of-fit 0.055 2 0.027 20.033 0.047Pure error 0.002 2 0.001Total (corr.) 10.476 10 1.164R2 0.994

ig. 1. Reduced viscosity as a function of FucoPol concentration in salt-free solutiona) and determination of the intrinsic viscosity in 0.1 M NaCl using the Huggins (�)nd Kraemer (�) equations (b).

olution, showing a non-linear dependence of these two param-ters. As the polymer concentration decreased, there was aecline of the reduced viscosity until a polymer concentrationf 0.02 g/L, followed by a steep increase for concentration val-es below 0.02 g/L. This behavior is usually perceived at low

onic strength aqueous solutions of polyelectrolytes [17], beingttributed to repulsive forces occurring between intra-chain groupsith the same charge, which leads to an increase of the moleculesydrodynamic volume, and consequently, to an increase of the

ntrinsic viscosity. As the biopolymer concentration increases,nter-chain interactions prevail over intra-chain repulsive forces,ecoming dominant at higher concentration values, leading to theeduced viscosity-concentration dependence normally observedor uncharged polymers [18].

The addition of ions to FucoPol solution leads to polysaccharide’segative charges shielding and disabling of intra-chain repulsions.s a consequence, when the measurements were performed in

he presence of salt (0.1 M NaCl), at 25 ◦C and pH 5.6 ± 0.05 (pHfter FucoPol dissolution, without any pH adjustment), linear plotsor Huggins and Kraemer extrapolations were obtained (Fig. 1b),nabling the determination of the intrinsic viscosity. The valuebtained was 8.86 ± 0.09 dL/g, which is in accordance with the pre-iously reported value, 8.9 dL/g [13], and also within the valueseferred in the literature for commercial polysaccharides, such asanthan, guar gum and Fucogel (5–50 dL/g) [19,20].

The Huggins constant (KH) determined for FucoPol was.58 ± 0.04, which is quite similar to that measured for Fucogel, aommercial fucose-containing bacterial polysaccharide (KH = 0.5520]). According to Morris et al. [21], KH should lie between 0.3 and.8, while values of KH higher than 1.0 are indicative of molecularggregation.

.1.2. Effect of pH and ionic strength on intrinsic viscosityTo get a better knowledge of FucoPol’s intrinsic viscosity, the

urface response methodology with a central composite rotat-ble design with two independent variables was performed. The

values of the independent variables, IS (M, NaCl) and pH, and ofthe dependent variable, [�], are listed in Table 1. For the cen-tral point runs (0.40 M NaCl and pH 7.0), the [�] achieved waswithin 8.23–8.30 dL/g, a value similar to the one obtained with-out pH adjustment (8.86 ± 0.09 dL/g, at 0.1 M NaCl and pH ≈ 5.6).Furthermore, results have also shown that within the wide rangeof ionic strengths and pH tested, [�] remained within 7.21 and8.54 dL/g. Only for the highest ionic strength (0.75 M NaCl; pH = 7)and highest pH (0.4 M NaCl; pH = 10.5) values tested, underintensive charge shielding, the intrinsic viscosity demonstrated apronounced decrease to 5.65 and 5.51 dL/g, respectively.

Statistical analysis was used to evaluate the significance of ionicstrength (M NaCl) and pH effect and their interactions on thequadratic model used for describing [�]. An appropriate analysisof variance (ANOVA) of the second order model showed a good fit(R2 = 0.99) and a sum of squares (SS) of 10.476, with 10 degrees offreedom (Table 2) [22]. Despite that, there was evidence of lack-of fit (p < 0.05), which means that the error predicted by the modelwas above the error of the replicates. Such result could be explainedby the pure error (calculated by the replicas of the central point),which was close to zero, giving an artificial sense of model withlack of fit.

The linear (IS; pH), quadratic (IS.IS; pH.pH) and the interaction(IS.pH) effects of ionic strength (M NaCL) (X1) and pH (X2)) on [�] isdescribed in Table 2. The intrinsic viscosity was influenced by all theeffects of ionic strength and pH (linear, quadratic and interaction),for a significance level of 5% (p < 0.05).

Such correlation between response and independent variablescan be graphically illustrated by the 3D response surface plots(Fig. 2). It can be observed that the intrinsic viscosity was kept prac-tically unchanged for a wide range of ionic strength and pH values(0.05–0.50 M NaCl and 3.0–8.0, respectively). Withal, it decreasedfor combinations of ionic strength and pH above 0.50 M NaCl and8.0, respectively. Results from Fig. 2 seems to indicate that for ionicstrengths above 0.75 M NaCl, the intrinsic viscosity decreases forany value of pH tested between 3.5 and 10.

The reduction of [�] values with the increase of ionic strength iscommon to other polysaccharides solutions with a polyelectrolytebehavior, such as colanic acid, for which the intrinsic viscositydecreases from 47.5 to 22.7 dL/g when the ionic strength increasesfrom 0.002 to 0.2 M NaCl [23], and also for the exopolysaccha-ride produced by Pseudomonas oleovorans, for which the intrinsicviscosity decreases from 14.0 to 4.9 dL/g when the ionic strengthincreases from 0.01 to 0.5 M NaCl [24]. With a higher ionic strengththere’s a strong polymer charge shielding which decreases theintramolecular repulsion and leads to a lower hydrodynamic vol-ume of the polymer molecule [24,25]. The difference is that, for

FucoPol, a substantial decrease of the intrinsic viscosity takes placeonly at higher values of ionic strength (>0.5 M NaCl).

614 C.A.V. Torres et al. / International Journal of Biological Macromolecules 79 (2015) 611–617

Fs

3

3

satspthoser

t�

F((

Table 3Cross model parameters estimated for different FucoPol concentrations.

FucoPol (wt.%) Cross model

�0 (Pa s) � (s) m

0.20 0.011 ± 0.000 0.005 ± 0.000 0.825 ± 0.0920.35 0.054 ± 0.006 0.028 ± 0.012 0.613 ± 0.0580.45 0.074 ± 0.002 0.016 ± 0.002 0.634 ± 0.0280.50 0.096 ± 0.004 0.022 ± 0.004 0.693 ± 0.0460.60 0.134 ± 0.008 0.033 ± 0.007 0.735 ± 0.0660.80 0.439 ± 0.038 0.098 ± 0.018 0.677 ± 0.0360.90 0.759 ± 0.024 0.159 ± 0.018 0.707 ± 0.0301.00 1.095 ± 0.036 0.282 ± 0.036 0.645 ± 0.0241.20 2.465 ± 0.066 0.553 ± 0.052 0.666 ± 0.016

1

ig. 2. Response surface plot of intrinsic viscosity [�] as a function of pH and ionictrength.

.2. Steady shear measurements

.2.1. Effect of polymer concentration on apparent viscosityThe effect of polymer concentration (0.2–1.2 wt.%) on the steady

hear behavior of FucoPol aqueous solutions was evaluated. Thepparent viscosity increased with increasing biopolymer concen-ration and, for all the concentration values studied, flow curveshowed a shear-thinning behavior, approaching a Newtonianlateau at lower shear rates (Fig. 3). This behavior is typical of solu-ions composed of entangled macromolecules [21,23,26,27] and itad already been observed in a previous work focused on the effectf temperature on the rheological properties of 1.0 wt.% FucoPololutions [15]. In addition, for all concentrations studied, the appar-nt viscosity did not change when reducing the applied shear rate,ight after shearing the sample up to a shear rate of 700 s−1.

The flow curves were fitted to the Cross model (Eq. (4)) usingTM ®

he software package Scientist from MicroMath , assuming

» �∞ and �0 » �∞, often used to describe flow curves presenting a

ig. 3. Shear rate dependence of viscosity for different concentrations of FucoPol.�) 0.20 wt.%; (©) 0.35 wt%; (�) 0.45 wt.%; (*) 0.50 wt.%; (♦) 0.60 wt.%; (�) 0.80 wt.%;×) 0.90 wt.%; (�) 1.0 wt.%; (�) 1.2 wt.%. Lines represented the fitted Cross equation.

RE =∑n

i=1(|xexp,i − xcalc,i|/xexp)/n is between 0.002 and 0.070.

Newtonian plateau at low shear rates, followed by a shear thinningregion [23,28]:

� − �∞�o − �∞

= 11 + (� �)m (4)

where � is the shear rate (s−1), � is the apparent viscosity (Pa s),�0 is the viscosity of the first Newtonian plateau (Pa s), �0 is theviscosity of the second Newtonian plateau (Pa s), � is a relaxationtime (s) and m is a dimensionless constant, which may be relatedto the exponent of the power law (n) by m = 1 − n.

Eq. (4) was fitted to the experimental data (Fig. 3) and theestimated parameter values are summarized in Table 3. The timeconstant � increases as FucoPol concentration increases. This factmeans that the relaxation time of the sample is higher due tothe higher overall number of entanglements established as morebiopolymer molecules are present in solution. Hence, the criticalshear rate value, i.e. the value corresponding to the transition fromNewtonian to shear-thinning behavior, which corresponds to thereciprocal of �, decreases as the polymer concentration increases.

Flow curves depicted in Fig. 3 were overlaid by scaling vertically,dividing by the respective viscosity of the first Newtonian plateau(�0), and horizontally, multiplying by the relaxation time (�), whichgenerated a master curve (Fig. 4). Such approach enables to under-stand FucoPol’s behavior for a wider range of shear rates. It wasemployed successfully for diverse polysaccharide [21,24,29].

With the application of the generalized equation:

�/�0 =1 + (� �)m (5)

Fig. 4. Master curve obtained shifting vertically dividing by �0, and horizontallymultiplying by relaxation time (�). Inset: � as function of FucoPol concentration.

C.A.V. Torres et al. / International Journal of Biological Macromolecules 79 (2015) 611–617 615

Fig. 5. Effect of ionic strength (M NaCl) and pH on FucoPol solutions’ viscositydependency on shear rate (1 wt.% FucoPol). (�) 0.15 M NaCl – pH 4.5; (�) 0.65 MNaCl – pH 4.5; (�) 0.15 M NaCl – pH 9.5; (×) 0.65 M NaCl – pH 9.5; (*) 0.40 M NaCl–71

aacsicriwecoi

3

omaFovnw

t(waaw–r

s�((lqiqe

b

Table 4Analysis of variance of the second order model for parameter �0 (viscosity of thefirst Newtonian plateau estimated by the Cross model).

Source of variation �0

Sum ofsquares

Df Meansquared

F-value p-value

IS 0.001 1 0.001 0.018 0.899pH 0.384 1 0.384 9.344 0.038IS.IS 0.480 1 0.480 11.687 0.027pH.pH 0.265 1 0.265 6.446 0.064IS.pH 0.001 1 0.001 0.035 0.860Lack-of-fit 0.163 2 0.054 37.209 0.120Pure error 0.001 2 0.001

curve has three distinct linear zones, characterized by differentslopes, and separated by two critical concentrations (c* and c**).This type of correlation is analogous to the one exhibited by otherhigh molecular weight microbial polysaccharides, such as xanthan

pH 7.0; (�) 0.40 M NaCl – pH 7.0; (+)0.40 M NaCl – pH 7.0; (�) 0.05 M NaCl – pH.00; (�) 0.75 M NaCl – pH 7.00; (♦) 0.40 M NaCl – pH 3.47; (�) 0.40 M NaCl – pH0.54. Lines represent the fitted Cross equation.

value of m = 0.681 ± 0.008 was obtained. This value is inccordance with the one presented by Morris, m = 0.76, for polysac-harides presenting strong interactions between polymer chains,uch as hydrogen bonds [30]. The concentration dependence of � isllustrated in the inset of Fig. 3. The solid line suggests � ∼ c4.12 foroncentrations above 0.6 g/dL, with an exponent near 4, which iseferred in the literature for polysaccharides exhibiting a strongnter-chain association [21,31]. By the contrary, polysaccharides

ith linear chains revealing no interchain association presentedxponent values of 2.08 [32]. Therefore, we may infer that, for theoncentration range studied (0.2–1.2 wt.%), FucoPol presents a rhe-logical behavior of a macromolecular entangled solution with highnter-chain association.

.2.2. Effect of ionic strength and pH on the apparent viscosityThe effect of pH and ionic strength on the steady shear behavior

f FucoPol solutions was also studied using the response surfaceethodology coupled with a central composite rotatable design,

ccording to Table 1. The study was carried out with a constantucoPol concentration of 1.0 wt.%. Fig. 5 presents the flow curvesf FucoPol aqueous solutions with different ionic strength and pHalues. The results show that all solutions presented a shear thin-ing behavior, as in the studies where the effect of concentrationas evaluated.

The flow curves were fitted by Eq. 4, which enabled the estima-ion of �0 values, presented in Table 1. For the central point runs0.40 M NaCl and pH 7.0), �0 achieved the highest value (1.20 Pa s),hich was similar to the one obtained for the 1.0 wt.% FucoPol

queous solution without pH adjustment (1.10 Pa s, at 0.1 M NaClnd pH ∼5.6). The lowest values were obtained for the solutionsith 0.40 M NaCl – pH 10.54, 0.15 M NaCl – pH 9.5 and 0.65 M NaCl

pH 9.5, with zero-shear viscosities (�0) of 0.37, 0.45 and 0.43 Pa s,espectively.

Statistical analysis was also used to evaluate the impact of ionictrength NaCl (M) and pH on the quadratic model for describing0. ANOVA of the second order model showed a satisfactory fitR2 = 0.85) (according to Lundstedt et al. [16]), a sum of squaresSS) of 1.109, with 10 degrees of freedom, and a non-significantack-of fit (p = 0.120) (Table 4). Table 4 shows the linear (X1; X2),uadratic (X1X1; X2X2) and the interaction (X1X2) coefficients of

onic strength (M NaCl) (X1) and pH (X2) on �0. Linear pH and

uadratic ionic strength are the factors which had a significantffect on �0 (p < 0.05).

The 3D response surface plot (Fig. 6) evidenced the correlationetween �0 and the independent variables pH and ionic strength.

Total (corr.) 1.109 10 0.123R2 0.850

The quadratic effect demonstrated that for the higher and lowerionic strength and pH values studied (axial points) the zero-shearviscosity decreased. It presented higher values (0.88 < �0 < 1.20) forionic strength and pH within 0.20–0.60 M NaCl and 3.0–8.0, respec-tively, corresponding to the ranges for which a higher intrinsicviscosity was observed (Fig. 2), i.e. where no significant shieldingfrom the ions in solution is expected.

3.3. Concentration regimes

The concentration regimes are generally obtained from theconcentration dependence of zero shear-rate specific viscosity(�sp,0) with biopolymer concentration, an approach used by sev-eral authors [18,20,33–35]. In this study, for concentration valuesbelow 0.1 wt.%, �sp,0 values were determined from the capillary vis-cometry data used to calculate the intrinsic viscosity in section3.1.1. For concentration values above 0.1 wt.%, �sp,0 was calcu-lated using the �0 values estimated in section 3.2.1 and presentedin Table 3. Fig. 7 displays the concentration dependence of zeroshear-rate specific viscosity (�sp,0) with FucoPol concentration. The

Fig. 6. Response surface of zero-shear viscosity (�0) as a function of pH and ionicstrength.

616 C.A.V. Torres et al. / International Journal of Biolo

Fp

[[[

monoiatiooeasrewcpt

4

saiispmvp

A

nPCaS

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[27] F. Chenlo, R. Moreira, C. Silva, Steady-shear flow of semidilute guar gum solu-

ig. 7. Concentration dependence of zero-shear specific viscosity for FucoPol sam-les (0.1 M NaCl; pH = 5.6).

36], the galactose-rich polysaccharide produced by P. oleovorans24] and the exopolysaccharide produced by P. acidi-propionici29].

The dilute regime is characterized by isolated polymerolecules with a free mobility and a negligible influence on each

ther. c* sets the dilute regime boundary, marking the onset of sig-ificant molecules overlapping [29]. The overlap parameter (C∗[�]),r space occupancy, was found to be approximately 0.8. Withncreasing concentration, polymer molecules start being directlyffected by each other [19]. This leads to a change from one stageo another, thus resulting in a significant increase of the slope. Thentermediate concentration region has a slope of 2.43, with spaceccupancy between 0.8 and 5.34. c** corresponds to the beginningf the entangled regime, where molecules interact intensively withach other [37]. The estimated values (c* ∼ 0.09 g/dL, c** ∼ 0.6 g/dL)re higher than the ones obtained for xanthan at the same ionictrength (c* ∼ 0.024 g/dL, c** ∼ 0.092 g/dL) [36]. This fact may beelated to a smaller hydrodynamic volume of FucoPol molecules,xpressed by its lower intrinsic viscosity ([�] = 8.86 ± 0.09 dL/g),hen compared to that of xanthan ([�] = 47.5 dL/g). The slope above

** (3.78) is in the range (3.3–4.0) presented for several random coilolymers in the literature (alginate, xanthan, colanic acid, galac-omannans) [21,23,38,39].

. Conclusions

This work showed that FucoPol produces viscous solutions withhear-thinning behavior at different polymer concentrations. Being

polyelectrolyte, its solution properties are dependent on pH andonic strength. Though, it presents intrinsic and apparent viscos-ty values with little variation within a wide range of pH and ionictrength values. From the results obtained, FucoPol shows a greatotential to be used as thickening agent in diverse aqueous for-ulations prepared with a wide range of pH and ionic strength

alues, to be used in applications such as oil drilling fluids, paints,harmaceuticals, cosmetics and food products.

cknowledgements

This work was supported by Fundac ão para a Ciência e a Tec-ologia (FC&T, Portugal) through projects UID/Multi/04378/2013,

Est-OE/AGR/UI0245/2014 and PTDC/AGR-ALI/114706/2009.ristiana A.V. Torres, Filomena Freitas and Ana R.V. Ferreiracknowledge FC&T for fellowships SFRH/BPD/87774/2012,FRH/BPD/72280/2010 and SFRH/BD/79101/2011, respectively.

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gical Macromolecules 79 (2015) 611–617

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